Humanization of Monoclonal Antibodies Targeting Alpha-Enolase of Candida Albicans and Their Potential Applications

In present, there are 3 types of isoforms of enolase molecule reported including a-enolase, g-enalose and b-enolase. a-enolase (ENO1) was first identified as a key component of glycolytic pathway and found to exhibit multiple biological functions with enzymatic and plasminogen-binding activities. In addition to its roles during the tumorigenesis, a-enolase molecule was recently demonstrated to be tightly associated with many diseases including autoimmune disorders and microbial infections. Candida albicans (C. albicans) is an opportunistic human pathogen, which colonizes at several sites including skin, oral, gastrointestinal track and vagina. C. albicans is also a major pathogen responsible for 50.4% of candidemia in hospitals, which occurs when Candida yeasts enter the bloodstream and is rarely seen in healthy people. As noticed, the cases with the infection of C. albicans have been dramatically increased in clinical patients with defective immunological functions. It was shown the α-enolase of C. albicans is expressed in a significant level on the cell wall and plays an crucial role during candidiasis. However, the pathogenesis of candidiasis is not exactly known at present. In this context, the previous studies have led us to hypothesize that the antigenic epitopes exposed on CaENO1 protein expressed on the surface of C. albicans represent important targets for the development of diagnostic agents and therapeutic drugs for clinical applications. Our preliminary studies have allowed us to generate and partially characterize an anti-CaENO1 antibody containing single chain variable fragment (scFv) using phage display antibody technology. Accordingly, in the present work, we propose to complete the objectives listed below. 1. Intensive screening of specific phages with anti-CaENO1 activity. 2. Binding and characterization of anti-CaENO1 scFv antibodies to the ENO1 expressed on cell surface of C.albicans. 3. Functional analysis of anti-CaENO1 scFv antibodies on mice infected with C.albicans. 4. Analysis and humanization of the anti-CaENO1 scFv antibodies. 5. Biological effect of the humanized anti-CaENO1 scFv antibodies on cell growth, hyphal formation and candidiasis animal model. 6. Engineering the humanized anti-CaENO1 scFv antibodies into whole IgG antibodies with Fc portion and re-examine their biological effects as above. The basis resulting from this proposed study will provide us additional support to unveil the specificity, toxicity, inhibitory activity and serum stability of these anti-CaENO1 antibodies for the development of diagnostic agents and therapeutic drugs against C. albicans infection in the future. For example, they could be applicable to detect C. albicans and C. tropicalis, which are 2 species of Candida mostly encountered in patients with candidiasis. Moreover, they also provide an alternative assessment to replace the blood bottle culture available in clinic. As for therapeutic application, they may be able to overtake the problems of antibiotic-resistance often seen in the patients with candidiasis.